In electrophoresis, an ionic sample is placed at one end of the column. The ionized components migrate differentially according to charge and bulk under the influence of an axially applied electric field. After a predetermined time, the electric field is removed and the components analyzed according to axial position along the tube.
Capillary zone electrophoresis has proven useful as an efficient method for the separation of certain small solutes. Attractive factors for CZE include the small sample sizes, little or no sample pretreatment, high solution, automation, and the potential for quantification and recovery of biologically-active samples. Detection and quantitation of the migrating ions can be carried out by measuring, for example, UV absorbance at a particular wavelength. Collection can be made directly into any conducting solution. Microcapillary gel electrophoresis is particularly wellsuited for separating proteins and other biopolymers. Very high efficiency and resolving power are possible with capillary gel columns, permitting rapid separation of biopolymers.
The columns of gel can be prepared by filling a tube with an aqueous mixture of acrylamide monomer, and then polymerizing the monomer. In the case of acrylamide, as is generally true in polymer chemistry, the polymer is substantially denser than the original prepolymer, e.g., the monomer, dimer, or oligomer, from which the polymer is formed. Accordingly, significant shrinkage occurs during polymerization. Besides crosslinked polyacrylamide gels, agarose is often employed as the medium in gel electrophoresis, especially for separation by molecular weight of large macromolecules such as nucleic acids. Agarose is a natural polysaccharide isolated from agar and agarose gel is a relatively transparent anticonvection medium that prevents broadening of the zones during separation. However, as in the formation of polyacrylamide gels, agarose gels are also adversely affected by shrinkage during polymerization.
As a consequence of this shrinkage, the forming gel has a tendency to pull away from the interior walls of the tube. The voids thus formed between the tube and the gel can disturb the uniformity of an applied electric field, seriously diminish the resolution of the electrophoresis process, and cause local heating. Moreover, the separation of the gel from the tube aggravates a tendency of the gel to migrate out of the tube during electrophoresis. Furthermore, the gel is not homogeneous, in terms of pore size, both radially (center to wall) and longitudinally (end to end).
One common approach with regard to the problem of maintaining the structural integrity of the gel during electrophoresis has been to coat the interior of the tube with a bonding agent which forms covalent bonds between the surface of the tube and the polymer chains. In these microcolumns, a bifunctional silane, for instance, is used as a bonding agent such that one functionality reacts with the inner surface of the capillary and the other (acrylic) functionality reacts with the polymerizing acrylamide monomer network, thus immobilizing the gel matrix. Although separation and resulting migration are mitigated, the tension introduced by the tendency of the gel to shrink during polymerization causes bubble-like voids within the gel itself. This adversely affects the gel's uniformity. These internal voids also distort the applied electric field and diminish the resolution of the electrophoresis process. See Karger et al., U.S. Pat. No. 4,865,706.
In a modification of the above approach, microcapillaries are prepared in which the polymeric gel contains hydrophilic polymers. However, the use of the hydrophilic polymer often leads to polymeric gels that are not sufficiently transparent because of phase separation during polymerization. See Karger et al., U.S. Pat. No. 4,865,707, issued Sep. 12, 1989.
The problem of shrinkage has been addressed and partially solved by pressurizing the monomer to its final polymer volume. See Bente, III et al., U.S. Pat. No. 4,810,456, issued Mar. 7, 1989. Use of pressure polymerization results in a fairly homogenous gel (as viewed under a microscope) but the gel develops "air cores" when it is exposed to a moderate electric field (100 V/cm). It is believed that the inhomogeneities which form during polymerization under pressure are caused by the rapid initiation of polymerization of the reaction due to the more reactive, vis-a-vis acrylamide, acrylic functional group near the wall surface. As the polymerization reaction progresses towards the center of the capillary, acrylamide monomer concentration in the middle of the capillary is decreased, resulting in an inhomogeneous gel.